Njapdounke et al., Afr J Tradit Complement Altern Med.

(2016) 13(4):1-7
doi:10.21010/ajtcam.v13i4.1

ANXIOLYTIC - LIKE PROPERTIES OF Hallea ciliata IN MICE

Njapdounke Kameni Jacqueline Stephanie1, Nkantchoua Nkamguie Gisele1, Moto Okomolo Fleur Clarisse2,
1
Taiwe Sotoing Germain3, Sidiki Neteydji , Pale Simon3, Ayissi Mbomo Espoir Rigobert2 ,Ngo Bum Elisabeth4
1
Department of Biological Sciences, Faculty of Science, University of Ngaoundere; Ngaoundere, Cameroon,
2
Department of Biological Science, High Training School, University of Yaounde I, Yaounde, Cameroon,
3
Department of Zoology and Animal Physiology- Faculty of Science, University of Buea, Buea, Cameroon, 4Institute
of Mines and Petroleum Industries, University of Maroua, Cameroon.

Corresponding author: Prof. Elisabeth Ngo Bum E-mail: [email protected]

Abstract

Background: The aim of the present study was to evaluate the anxiolytic properties of the decoction of stem bark of Hallea ciliate in mice.
The decoction of Hallea ciliata is used in traditional medicine in Cameroon to treat diseases like anxiety disorders, fever, infantile
convulsions and malaria.
Materials and Methods: Stress induced hyperthermia, elevated plus maze, open field and hole-board tests were used. Four different doses
of the decoction were administered to mice and their effects were compared to the effects of diazepam and vehicle. Phytochemical
characterization of the decoction revealed the presence of alkaloids, flavonoids, tannins and saponins.
Results: Administration of Hallea ciliata resulted in a significant decrease of stress induced hyperthermia in mice at the doses of 29.5, 59
and 118 mg/kg. In the elevated plus maze test, Hallea ciliata increased the number of entries and the percentages of entries and time into
the open arms, and reduced the number of entries and the percentages of entries and time into the closed arms. In the hole-board test,
Hallea ciliata increased the number of both head-dipping and crossing and decreased the latency to the first head-dips and rearing. The
decoction of Hallea ciliata and diazepam increased locomotion in the open field test.
Conclusion: The number of rearing and the mass of fecal boli produced were decreased in mice treated with decoction and
diazepam. In conclusion, the results indicated that decoction of Hallea ciliata has anxiolytic-like properties in mice and could
potentially be used for anxiety treatment.

Key words: anxiety, herbs, pharmacology, diazepam.

Introduction
Anxiety and stress disorders are currently among the ten most important public health concerns, according to the World
Health Organization and, have reached epidemic proportions (Thase, 2006). In Cameroon, anxiety disorders are among the top three mental
diseases (Ministry of Health of Cameroon, 2006). These disorders are recognized as main risk factors for many other diseases,
including cardiovascular, metabolic and neuropsychiatric diseases (Cryan and Holmes, 2005; Thase, 2006). Furthermore, anxiety is among
the most prevalent mental disorder with very high comorbidity and severe impact on quality of life (Ngo Bum et al., 2011). In Africa, and
particularly in Cameroon, patients mostly go to traditional healers because of the stigma of mental diseases and the side effects of anxiolytic
drugs (Abdallah et al., 2014; Griffiths et al., 2014).
A growing number of herbal medicines are being introduced into psychiatric practice on the basis of their efficacy and low side
effects for the treatment of psychiatric disorders, as severe depression, anxiety (Ji et al., 2006; Ngo Bum et al., 2009; Shaw et al., 2007;
Taiwe et al., 2010). Hallea ciliata Aubrey & Pelegr. (Rubiaceae) (H. ciliata) is used in several conditions in African traditional medicine,
particularly in the Northern Cameroon, to treat pain, infantile convulsions, anguish, inflammation, fever, arterial hypertension, diarrhea,
diabetes, parasitic infection, mental impairment, and as local antiseptic,. The decoction of this plant together with piper guinense and
Xylopia aettiopa is effective for lung diseases, thus being nontoxic in human, (FAO, 1986). Previous studies showed that H. ciliata
possess anti-inflammatory and analgesic properties (Dongmo et al., 2003); myorelaxant, antiplasmodial and antipyretic properties
(Mibindzou, 2004); anti- trypanosomiase activity (Ogbunugofor et al., 2007) and is a good immunostimulator (Fofana, 2005).
However, very little information is available on the effects of H. ciliata on the central nervous system. The present study was undertaken
to verify the hypothesis that H. ciliata could exert anxiolytic properties because of its used in traditional medicine in Cameroon to treat
anxieties disorders.

Material and Methods

Animals and Treatment

Adult male and female Swiss mice (Mus musculus) weighting 20-24 g were used. The animals were bred in the colony
established at the department of Biological Sciences of the University of Ngaoundere (Cameroon). They were housed 5 or 6 per cage
and were familiarized with the experimenters and the laboratory environment for at least 4 days before the experiments. Food and
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 Njapdounke et al., Afr J Tradit Complement Altern Med. (2016) 13(4):1-7
doi:10.21010/ajtcam.v13i4.1
water were available ad libitum.

The animal room was maintained at 25 ± 1 °C with a 12/12h light/dark cycle (light on at 6.00 a.m).
All experiments were carried out in a quiet room under controlled light conditions between 10:00 a.m. and 3:00 p.m. Mice were
divided randomly in 6 groups: one negative control group received distilled water (vehicle), one positive control group received a
well-known anxiolytic drug (diazepam or phenobarbital) depending on the test, and four test groups received different doses of the
decoction of H. ciliata.
All experiments were performed in accordance with the National Ethical Committee (N°.FWA-IRB00001954) and the
internationally accepted Principles for Laboratory Animal Use and Care presented in the US guidelines. All efforts were done to
minimize suffering and number of animals used.
Diazepam ampoules (HOFFMANN LA ROCHE, BASEL, SWITZERLAND) and phenobarbital (SIGMA-ALDRICH CO.,
ST. LOUIS, MO, USA) were used as reference drugs. All solutions were prepared freshly in distilled water on the test days and
administered intraperitoneally (i. p) in a volume of 10 mL/kg body weight.
Diazepam, phenobarbital and the different doses of the decoction of H. ciliata were administered orally (p.o) once, 1 hour
prior to the beginning of all behavioral tests.

Preparation of the Decoction

Stem bark of H. ciliata were collected in Dang, locality of Ngaoundere (Adamaoua, Cameroon). Voucher specimen of H.
ciliata was depDosited at the National Herbarium of Cameroon in Yaounde under the number 2101HNC. For the preparation of the
decoction, 5 g of H. ciliata stem bark were macerated in 50 mL of distilled water and boiled to 100 °C for 20 minutes. After cooling,
the mixture was filtered with wattman paper n° 1 and used for animal experiments. In other to quantify the dried extract of the
decoction, the obtained decoction was dried and the residue was weighed. The doses of the decoction were expressed in mg of dried
extract per kg of body weight. The decoction was prepared each day of the experiment. The following doses were used: 11.8, 29.5,
59 and 118 mg/kg.

Phytochemical Screening

Preliminary qualitative chemical screening of the decoction of the stem bark of H. ciliata was done using the methods
described for the determination of flavonoids, alkaloids, saponins, tannins, anthraquinons and polyphenols (Harbone, 1976).

Behavioral Tests

Behavioral recording was done blindly.

Stress Induced Hyperthermia (SIH) Test

Experiments were performed according to the methodology described by Borsini et al., (1989) with small modifications
(Ngo Bum et al., 2009). The animals (10 per group) were weighed and marked on the tail. Phenobarbital (20 mg/kg, i.p) was given
to positive control group, the four doses of the plant were given to the four test groups and distilled water as vehicle was given to the
negative control group. 1 hour after each treatment, the rectal temperature was recorded by introducing the rectal borer of
thermometer (2 mm of diameter and 2 cm of length) in the rectum of the mice. Rectal borer was put into saline solution (NaCl 9ooo / )
before each temperature measurement. SIH was determined as the difference between the mean temperature of three last mice and
the mean temperature of the three first mice in each group.

Elevated Plus Maze (EPM) Test

This test is based on the natural empty space fear of rodents (Ollat and Pirot, 2003). The maze made of wooden consisted
of two open arm (15 cm x 45 cm) and two closed arms (15 cm x 45 cm x 20 cm), extending from a central platform (5 cm x 5 cm)
and elevated to a height of 50 cm above the floor (Ngo Bum et al., 2009). Here, diazepam (3 mg/kg, i.p.) was given as positive
control. One hour after treatment, each animal was placed individually in the center of the maze facing an open arm, away from the
observer. The behaviors of the animals were recorded for 5 minutes. The number of entries and the time spent into the open and
closed arms were registered. An entry was defined as entry of head and at least the half of the body of animal into an arm. The
percentage of time spent into open or closed arms (open or closed arm time/5 min x 100) and percentage of entries (open or closed
arm entries/total entries x 100) were calculated for each animal. The number of rearing, head-dipping and grooming were recorded
(Augustsson, 2004). The maze was cleaned with ethylic alcohol 70° after each mouse test.

Hole-Board (HB) Test

The hole-board apparatus consisted of a wooden box (40 cm x 40 cm, 1.8 cm height). The floor was divided in 16

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doi:10.21010/ajtcam.v13i4.1
equidistant holes of 3 cm in diameter (Lourenzo et al., 2001). The positive control group received diazepam (0.5 mg/kg, i.p.) and four
tests groups receive different doses of H. ciliata decoction (11.8, 29.5, 59 and 118 mg/kg, p.o.). One hour after treatment each animal was
placed individually in the center of the board facing away the observer. And the number of rearing (when the animal stand on its back
paws, raises its forepaws off the ground, extending its body vertically), head-dipping (when the animal places its head into one of the
holes), crossing (when the animal enter a new area with all four paw) were recorded for 5 min. The latency to the first head-dipping was
measured using a stopwatch (Taiwe et al., 2010). The floor of the apparatus was cleaned with 70 % alcohol solution between each trial.

Open Field (OF) Test

This method is used to evaluate locomotor activity, level of exploration and emotional reaction of animals (Crawley,
1985). Open field consisted of a surrounding square (40 cm x 40 cm) divided in 16 small square and 1 center field (10 cm x 10 cm) with a
wall of 19 cm high (Brown et al., 2007; Jenck et al., 1997). The positive control group received diazepam (0.3 mg/kg, i.p.). One hour after
treatment, each mouse was placed individually in the center of the arena and the number of crossing, grooming, rearing, the time spent
in the center and fecal boli weight were recorded for 5 min. Recording were measured manually with a stopwatch.

Data Analysis

Comparisons of the results were performed using computerized linear regression analysis, of GraphPad Prism (version
4.00, GraphPad Soft- ware Inc., San Diego, CA, USA). The statistical analysis of the data was performed in each graph by one-way
analysis of variance (ANOVA) followed by Dunnet’s bilateral or multiple comparison test. In all cases differences were considered
significant at p ≤ 0.05.

Results
Phytochemical Screening

Chemical screening of the decoction of stem bark of H. ciliata showed the presence of polyphenols (flavonoids, tannins,
phlobotannins), alkaloids, saponins, and anthraquinons.

Effect of H. Ciliata on Stress Induced Hyperthermia Test

The decoction of H. ciliata reduced dose dependently the SIH from 2.27 °C in the control group to -0.4° C at the dose of
118 mg/kg [F (6, 60) = 0.45; P<0.01]. HIS was also reduced by phenobarbital (Fig 1). In the contrary, H. ciliata had no effect on the
body temperature of mice (Fig 2).

Figure 1: Effect of the decoction of H. ciliata on the stress induced hyperthermia

Data are ∆°C (the difference between the mean temperature of the three last mice and the mean temperature of the three first mice).
N = 6, p ≤ 0.01, bp ≤ 0.001;c one-way analysis of variance (ANOVA) followed by Dunnet’s bilateral comparison test. CON =
distilled water (vehicle), PHO = phenobarbital 20 mg/kg. All other groups were compared to CON, the negative control group

Figure 2: Effect of the decoction of H. ciliata on the body temperature on stress induced hyperthermia in mice.
b
Data are temperature (°C). N = 10, p ≤ 0.01; one-way analysis of variance (ANOVA) followed by Dunnet’s bilateral comparison
test. CON = distilled water (vehicle), PHO = phenobarbital 20 mg/kg. All other groups were compared to CON, the negative control
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 Njapdounke et al., Afr J Tradit Complement Altern Med. (2016) 13(4):1-7
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group.

Effect of H. Ciliata on Elevated plus Maze

The administration of the decoction resulted in a significant increase in the number of entries into open arms from 1.67 in
the negative control group to 9.67 at the dose of 118 mg/ kg of H. ciliata [F (6, 35) = 9.13; p < 0.001] (Table 1). The percentage of
open entries was therefore highly significantly increased from 24.54% in the control group to 76.82% at the dose of 118 mg/ kg of
H. ciliata [F (6, 35) = 19.82; p < 0.001]. The percentage of time spent in the open arms increased also from 7.17% in the control
group to 58.00% [F (6, 35) = 101.46; p < 0.001] at the dose of 118 mg/kg of H. ciliata. As expected, in the positive control group
3 which received diazepam (3 mg/kg, i.p.), the percentage of entries and time spent into the open arms increased (Fig 3). The time
spent in the closed arms was as well reduced by both the decoction and diazepam. Diazepam and the decoction (dose 118 mg/kg)
also induced a significant reduction of percentage of entries in the closed arms, from 73.94% in the vehicle-treated group to 23.18%
and 16.59% [F (6, 35) = 19.78; p< 0.001], respectively (Fig 4). The number of rearing [F (6, 35) = 8.55; p< 0.001], head-dipping (F
(6, 35) = 16.85; p< 0.0001] and stretched attend posture [F (6, 35) = 3.93; p < 0.008] were also reduced by diazepam and the
decoction while the number of grooming increased (Table 1).

Figure 3: Effect of the decoction of H. ciliata on EPM in mice.

Data are the percentage of open arms entries and the percentage of open arms time. N = 6; one-way analysis of variance (ANOVA)
followed by Dunnet’s multiple comparison test. %OE = Percentage of open arm entries, %OT = Percentage of time spent in open
arm, CON = distilled water (vehicle), DZP = diazepam 3 mg/kg.

Figure 4: Effect of the decoction of H. ciliata on EPM in mice

Data are the percentage of closed arms entries and the percentage of closed arms time. N = 6; one-way analysis of variance
(ANOVA) followed by Dunnet’s multiple comparison test. %CO = Percentage of closed arms entries, %CT: Percentage of time
spent in closed arms, CON =distilled water (vehicle), DZP = diazepam 3 mg/kg.

Table 1: The number of open arms entries, closed arms entries, rearing, head- dipping and grooming on Elevated Plus Maze

Doses of H. ciliate Distilled 11.8 29.5 59 118 Diazepam

(mg/kg) water
Open arms entries 1.67 ± 0.44 7.83 ± 0.89 c 8.00 ± 0.67c 9.00 ± 2.67c 9.67 ± 2.67 c 10.00 ± 1.33 c
Closed arms entries 6.00 ± 2.67 4.00 ± 0.67 3.83 ± 1.5 a 3.16 ± 0.55b 2.67 ± 0.67b 2.00 ± 0.33c
Total arms entries 7.67 ± 2.83 11.83 ±1.16a 11.83 ± 1.67a 12.17 ±2.83 a 12.33 ± 2.33a 12.00 ± 1.33 a
c c c c
Rearing 11.67 ± 2.09 6.50 ± 1.28 5.50 ± 1.50 6.83 ± 1.89 5.50 ± 2.17 5.83 ± 0.89c
Head- dipping 12.17 ± 1.22 5.33 ± 1.17c 4.83 ± 1.83c 4.50 ± 1.00c 3.17 ± 0.56 c 3.67 ± 1.00c
a
Grooming 1.67 ± 0.27 2.00 ± 0.67 2.16 ± 0.61 1.67 ± 0.89 3.00 ± 1.00 4.17 ± 1.60c
SAP 8.17 ± 4.22 8.16 ± 2.14 5.00 ± 2.67 4.67 ± 2.44 3.00 ± 3.00a 0.83 ± 0.56b
Data are mean ± S.E.M., n = 6, = ap < 0.05, =b p < 0.01, c = p < 0.001, ANOVA followed by Dunnett (bilateral) comparison test.
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Open Field

As in the elevated plus maze, the number of rearing was decreased both by diazepam and the decoction from 24.4 for the
vehicle- treated group to 12.0 at the dose 118 mg/kg of H. ciliate and to 4.4 for diazepam [F(5,29) = 9.82; p < 0.001] (Table 2). The
decoction and diazepam also decreased fecal boli [F (5, 29) = 9.81; p < 0.001]. Contrarily, the decoction increased the number of crossing
and center time from 20.8 and 3.4 in the vehicle-treated group to 72.4, and 16.6 at the dose of 118 mg/kg of H. ciliata, [F( 5,29) = 12.00; p
< 0.001] and [F (5,29) = 39.023; p < 0.001], respectively. Increase was also noted in grooming (Table 2).

Table 2: Number of crossing, rearing, grooming, center time and fecal boli on Open Field

Doses of H. ciliate Distilled 11.8 29.5 59 118 Diazepam

(mg/kg) water
Crossing 20.80 ± 2.24 58.40 ± 18.24b 80.40 ± 4.88 c 84.20 ± 6.24c 72.40 ± 10.32c 78.40 ± 9.68 c
Rearing 24.40 ± 7.92 13.60 ± 4.88b 13.00 ± 4.00 c
15.60 ± 5.12b 12.00 ± 4.40 c
4.40 ± 1.92 c
a
Grooming 0.60 ± 0.48 2.00 ± 0.80 2.60 ± 1.52 2.60 ± 1.12 3.00 ± 1.20 3.60 ± 0.88b
Center time (s) 3.40 ± 1.84 6.60 ± 5.52 10.40 ± 3.12 14.00 ± 10.00a 16.60 ± 3.52 a 82.00 ± 10.00 c
Fecal boli (g) 0.27 ± 0.22 0.001 ± 0.002c 0.001 ± 0.001 c
0.018 ± 0.003c 0.00 ± 0.00 c
0.00 ± 0.00 c
Data are mean ± S.E.M., n= 6, = ap < 0.05, =b p < 0.01, c = p < 0.001, ANOVA followed by Dunnett (bilateral) comparison test.

Hole-Board

The number of rearing was significantly reduced by H. ciliata, from 7.8 in the vehicle treated-group to 3.0 and 2.8 at the doses
29.5 and 59 mg/kg of the decoction, respectively. The latency time to the first head-dipping was also significantly reduced by H. ciliata,
from 8.6 in the vehicle-treated group [(F (5, 29) = 8.99; p < 0.001] to 4.0 (dose of 118 mg/kg of the plant) and to 2.0 (diazepam). In the
contrary, the number of head-dipping increased from 13.2 in the vehicle-treated group to 40.4 at the dose of 118 mg/kg of the decoction and
to 33.6 for the diazepam- treated group [F (5, 29) = 4.52; p < 0.005]. The number of grooming was also increased by the decoction [F (5,
29) = 0.36; p < 0.90] and diazepam (Table 3).

Table 3: Number of crossing, rearing, grooming, head-dipping and first head - dipping time on Hole Board

Doses of H. ciliate Distilled 11.8 29.5 59 118 Diazepam

(mg/kg) water
Crossing 28.00 ± 6.40 28.00 ± 6.40 42.80 ± 9.36 38.20 ± 7.28 19.00 ± 6.40 31.80 ± 15.78
Rearing 7.80 ± 2.64 3.00 ± 0.80 a 3.00 ± 2.40a 2.80 ± 0.72a 3.20 ± 1.76 a 5.80 ± 2.56
Grooming 0.60 ± 0.48 2.00 ± 0.80 2.60 ± 1.52 2.60 ± 1.12 3.00 ± 1.20 a 3.60 ± 0.88b
Head-dipping 13.20 ± 3.76 17.80 ± 4.56 20.00 ± 7.20 26.80 ± 3.84 40.40 ± 16.32 b 33.60 ± 9.92 a
Latency to 8.60 ± 2.07 8.00 ± 1.00 7.20 ± 1.48 5.00 ± 1.87a 4.00 ± 3.16b 2.00 ± 1.00c
first head-dipping (s)
Data are mean ± S.E.M., n= 6, = ap < 0.05, =b p < 0.01, c = p < 0.001, ANOVA followed by Dunnett (bilateral) comparison test.

Discussion
The anxiolytic properties of the decoction of H. ciliata were investigated for the first time using behavioral testing in mice.
The present results showed that the decoction of the stem bark of H. ciliata reduced hyperthermia induced by stress. This decrease similar
to that induced by phenobarbital, suggested anxiolytic properties of the plant, as hyperthermia induced by stress is reversed by anxiolytics
(Berend and al., 2003; Borsini et al., 2002; Lecci et al., 1990; Ngo Bum et al., 2009). Anxiolytic properties of the decoction were
confirmed in the elevated plus maze, the open field and hole board tests. The experiment on the elevated plus maze clearly showed that
vehicle-treated mice had a strong preference for the closed arms compared to those treated with the decoction of H. ciliata. The number of
entries and the time spent into open arms and their percentage; the main behavioral parameters of the evaluation of the anxiolytic effect
on this maze (Fakeye et al., 2008; Grundmann et al., 2007; Pollyanna et al., 2007; Rabbani et al., 2008; Reginatto et al., 2006) were
increased by the decoction of H. ciliata. Significant decrease in the number of "rearing" associated both to the decrease of closed arms
entries and the increase of open arms entries revealed that the increase in open arms entries was not due to the decoction of H. ciliata
induced locomotion but rather to the exploration confirming the decrease of anxiety in mice treated with the decoction (Ngo Bum et al.,
2009; 2011). In addition, the plant induced a significant increase in the number of grooming in mice treated with the highest dose of H.
ciliata (118 mg/kg) and diazepam. Grooming is a behavior that reflects the removal of stress in animals (Augustsson, 2004).
In open field test, H. ciliata and diazepam increased the number of "crossing". The increase in the number of crossing
(exploratory activity) is a sign of intrinsic inhibition of anxiety induction and not an increase in locomotion since rearing which is a
locomotion indicator in this test was reduced (Ngo Bum et al., 2009; Pitchaiah et al., 2008; Pollyanna and al., 2007). Reduction of
defecation in mice treated with diazepam and the extract of H. ciliata also suggested an anxiolytic activity (Shaw et al., 2007). In hole-
board test, the decoction of H. ciliata increased the number of crossing and head-dipping (exploratory activity) and decreased its latency
of onset. Since increasing the number of crossing and head-dipping in this test is a sign of anxiolysis (Crawley, 1985; File and Wardill.,
1975; Li Min et al., 2005; Lourenzo et al., 2001; Tsuji et al., 1998), these results also indicated the anxiolytic properties of H. ciliata. H.
ciliata could have exerted anxiolytic properties through its flavonoids, alkaloids and tannins contain. For instance flavonoids selectively

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 Njapdounke et al., Afr J Tradit Complement Altern Med. (2016) 13(4):1-7
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bind with high affinity to benzodiazepine receptors and induce a significant anxiolytic activity (Grundmann et al., 2007). As anxiolytic
activities could be mediated by different mechanism of action, we hypothesized that the anxiolytic properties of the decoction H. ciliata
could be due to the interaction of its contained compounds with receptors in the central nervous system such as benzodiazepine and GABA
sites of GABA-A receptor complex as agonists (EPM test is very sensitive to benzodiazepine receptor agonists), 5-HT1 receptor as agonists
(Belzung, 1999; Garcia-Garcia et al., 2014; Lolli et al., 2007; Rodgers et al., 1997) or 5-HT-2 and 5-HT-3 receptors as antagonists (Brown
et al., 2007; Ferrero et al., 1999; Rodgers, 2001).

Conclusion

The decoction of H. ciliata showed anxiolytic like-effects that are used in traditional medicine in Cameroon to treat
anxieties disorders.

Acknowledgements
The authors thank the Laboratory of Medicinal Plants, Health and Galenic Formulation of the Faculty of Science of the
University of Ngaoundere for the support and didactical equipment.

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